Engineered ligase variants
Inventors
Miller, Mathew G. • Vroom, Jonathan • Dellas, Nikki • Baskerville, Donald S. • Gomes, Sandy M. • Elgart, David • Viduya, Judy Victoria Antonio
Assignees
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Publication Number
US-12344872-B2
Publication Date
2025-07-01
Expiration Date
Abstract
The present invention provides engineered ligase polypeptides and compositions thereof, as well as polynucleotides encoding the engineered ligase polypeptides. The invention also provides methods for use of the compositions comprising the engineered ligase polypeptides for diagnostic and other purposes.
Core Innovation
The disclosure relates to engineered T4 DNA ligase variants defined by polypeptide sequences identified by SEQ ID NOs and by specific amino-acid substitution sets using position numbering relative to the cited SEQ IDs. In particular, the engineered T4 DNA ligase comprises a polypeptide sequence that consists of SEQ ID NO:38.
The patent describes improved DNA ligation performance, including ligation under low DNA substrate concentrations and in cell-free DNA. This is associated with reduced adapter dimer formation and lower adapter amounts, and the engineered ligases are described as outperforming wild-type T4 ligase.
The patent further describes improved physical and operational robustness of the engineered T4 DNA ligase, including increased thermostability, broader pH stability, PEG-free/low-crowder ligation, and improved inhibitor tolerance. The engineered ligases can enable faster ligation with fewer adapter dimers.
Claims Coverage
The provided content includes one independent claim directed to an engineered T4 DNA ligase defined by a specific polypeptide sequence. The claim scope is based on one inventive feature: the exact sequence definition of the ligase polypeptide.
Engineered T4 DNA ligase consisting of a specific SEQ ID NO
An engineered T4 DNA ligase comprising a polypeptide sequence, wherein the polypeptide sequence consists of SEQ ID NO:38.
Claim coverage centers on an engineered T4 DNA ligase whose polypeptide sequence is defined to consist of SEQ ID NO:38.
Stated Advantages
Improved ligation under low DNA substrate concentrations, including cell-free DNA.
Reduced adapter dimer formation by requiring lower adapter amounts.
Increased thermostability.
Broader pH stability.
PEG-free/low-crowder ligation.
Improved inhibitor tolerance.
Engineered ligases outperform wild-type T4 ligase.
Enables faster ligation with fewer adapter dimers.
Documented Applications
Ligation assays using labeled DNA substrates, including plate-capture ligation and capillary electrophoresis ligation assays [procedural detail omitted for safety].
Adapter dimerization selectivity profiling in ligation assays [procedural detail omitted for safety].
Temperature and pH activity profiling in ligation assays [procedural detail omitted for safety].
Conversion profiling versus enzyme/adapter concentration in ligation assays [procedural detail omitted for safety].
Ligation of cell-free human serum DNA substrates in ligation assays [procedural detail omitted for safety].
NGS library preparation.
Chromatin conformation capture (C3/3C/4C/5C/Hi-C/C3/3C-Hi-C).
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